Modification of extracellular matrix proteins by oxidants and electrophiles.

The extracellular matrix (ECM) is critical to biological architecture and determines cellular properties, function and activity. In many situations it is highly abundant, with collagens and elastin being some of the most abundant proteins in mammals. The ECM comprises of multiple different protein species and sugar polymers, with both different isoforms and post-translational modifications (PTMs) providing a large variety of microenvironments that play a key role in determining tissue structure and health. A number of the PTMs (e.g. cross-links) present in the ECM are critical to integrity and function, whereas others are deleterious to both ECM structure and associated cells. Modifications induced by reactive oxidants and electrophiles have been reported to accumulate in some ECM with increasing age. This accumulation can be exacerbated by disease, and in particular those associated with acute or chronic inflammation, obesity and diabetes. This is likely to be due to higher fluxes of modifying agents in these conditions. In this focused review, the role and effects of oxidants and other electrophiles on ECM are discussed, with a particular focus on the artery wall and atherosclerotic cardiovascular disease. Modifications generated on ECM components are reviewed, together with the effects of these species on cellular properties including adhesion, proliferation, migration, viability, metabolic activity, gene expression and phenotype. Increasing data indicates that ECM modifications are both prevalent in human and mammalian tissues and play an important role in disease development and progression.

The inflammatory oxidant peroxynitrous acid modulates the structure and function of the recombinant human V3 isoform of the extracellular matrix proteoglycan versican.

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO-/ONOOH. Both reagent ONOO-/ONOOH and SIN-1 (a thermal source of ONOO-/ONOOH) modified Tyr, Trp and Met residues. ONOO-/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO-/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO-/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

Peroxynitrous acid-modified extracellular matrix alters gene and protein expression in human coronary artery smooth muscle cells and induces a pro-inflammatory phenotype.

Leukocytes produce oxidants at inflammatory sites, including within the artery wall during the development of atherosclerosis. Developing lesions contain high numbers of activated leukocytes that generate reactive nitrogen species, including peroxynitrite/peroxynitrous acid (ONOO-/ONOOH), as evidenced by the presence of oxidized/nitrated molecules including extracellular matrix (ECM) proteins. ECM materials are critical for arterial wall integrity, function, and determine cell phenotype, with smooth muscle cells undergoing a phenotypic switch from quiescent/contractile to proliferative/synthetic during disease development. We hypothesized that ECM modification by ONOO-/ONOOH might drive this switch, and thereby potentially contribute to atherogenesis. ECM generated by primary human coronary artery smooth muscle cells (HCASMCs) was treated with increasing ONOO-/ONOOH concentrations (1-1000 µM). This generated significant damage on laminin, fibronectin and versican, and lower levels on collagens and glycosaminoglycans, together with the increasing concentrations of the damage biomarker 3-nitrotyrosine. Adhesion of naïve HCASMC to ECM modified by 1 µM ONOO-/ONOOH was enhanced, but significantly diminished by higher ONOO-/ONOOH treatment. Cell proliferation and metabolic activity were significantly enhanced by 100 µM ONOO-/ONOOH pre-treatment. These changes were accompanied by increased expression of genes involved in mitosis (PCNA, CCNA1, CCNB1), ECM (LAMA4, LAMB1, VCAN, FN1) and inflammation (IL-1B, IL-6, VCAM-1), and corresponding protein secretion (except VCAM-1) into the medium. These changes induced by modified ECM are consistent with HCASMC switching to a synthetic/proliferative/pro-inflammatory phenotype, together with ECM remodelling. These changes model those in atherosclerosis, suggesting a link between oxidant-modified ECM and disease progression, and highlight the potential of targeting oxidant generation as a therapeutic strategy.